The present invention relates to a composition, device and method for determining the presence or concentration of a peroxidatively active substance in a test sample. More particularly, the present invention relates to a new and improved method of assaying a liquid test sample such as urine for a peroxidatively active substance, e.g. occult blood, by utilizing a reduction resistant indicator reagent composition. The indicator reagent composition, in a wet phase assay or a dry phase assay, undergoes a detectable or measurable response upon contact with a test sample containing a peroxidatively active substance.
Peroxidase is an enzyme that catalyzes the oxidation of various compounds, such as phenols and amines, by peroxides. In addition, particular compounds have been termed pseudoperoxidases because they behave in a manner similar to the peroxidase enzyme. Pseudoperoxides liberate oxygen from hydroperoxides creating an oxidant capable of accepting an electron from a donor species. Accordingly, the pseudoperoxidases are enzyme like in that they catalyze, or otherwise participate in, reactions between peroxides and oxidizable compounds. The pseudoperoxidases, which include hemoglobin and its derivatives, are also termed peroxidatively active substances. For example, a peroxidatively active substance, such as hemoglobin and its derivatives, catalyzes the interaction between a hydroperoxide and an oxidizable dye. In such interactions, the peroxidatively active substance imitates the peroxidase enzyme and catalyzes or otherwise participates in an interaction between the oxidizable dye and the peroxide. The oxygen transferred from a peroxide to a peroxidatively active substance creates an oxidant capable of accepting an electron from an oxidizable dye. The resulting interaction provides a detectable response, such as a color transition, wherein the intensity of the response is indicative of the presence or the concentration of the peroxidatively active substance.
Assays for a peroxidatively active substance are based upon the above described chromogenic interaction, wherein the degree and intensity of the color transition of the indicator dye are correlated to the concentration of the peroxidatively active substance in the test sample. Assays for a peroxidatively active substance are particularly useful in detecting and measuring low concentrations of blood, often termed "occult" blood, in body fluid samples such as urine, feces or gastrointestinal contents. Although occult blood is not visible to the naked eye, its detection is important in the diagnosis of hemorrhages in the stomach, intestines and urinary tract. The hemorrhages are caused, for example, by tumors, ulcers or inflammations of the organ in question. Presently, most methods of determining the presence of occult blood in a test sample are based upon the pseudoperoxidase activity of hemoglobin.
Myoglobin, the red respiratory pigment of muscle tissue, is another peroxidatively active substance. Myoglobin is very similar to hemoglobin in its composition and chemical reactions. Myoglobin can be liberated from muscle cells by certain types of injury, and in such cases, the myoglobin will circulate in the plasma and be excreted in the urine. In addition, certain genetic muscle disorders can cause the muscles to lose myoglobin that subsequently appears in the urine. Myoglobin also is found in the urine after a cardiac infarct. Other peroxidatively active substances are also present in leukocytes and bacteria, and, in general, the detection of a peroxidatively active substance is especially important in the diagnosis of diseases and infections of the kidneys and urinary tract. Accordingly, accurate and thorough assays of urine and other test samples for peroxidatively active substances must be available for both laboratory and home use. The assays must permit the detection and measurement of the peroxidatively active substance such that a correct diagnosis can be made and correct medical treatment implemented, monitored and maintained.
It is advantageous for the assay method for a peroxidatively active substance to be suitable for use both in wet phase assays and in dry phase reagent strips for the rapid, economical and accurate determination of a peroxidatively active substance in urine or other test sample. Methods based on dip-and-read dry phase test strips have proven especially useful because dry phase test strip methods are readily automated and provide reproducible and accurate results. Some test strips used in assays for peroxidatively active substances have a single test area consisting of a small square pad of a suitable carrier matrix impregnated with an indicator reagent composition comprising an indicator chromogen, such as a benzidine dye; a hydroperoxide; and a buffer. The assay for a peroxidatively active substance in urine is performed by dipping the colorimetric test strip into a well mixed, uncentrifuged urine sample and then comparing the resulting color of the test area of the strip to a standardized color chart provided with the test strip container. Such occult blood tests are usually included on multideterminant reagent strips to screen urine samples during routine physical examinations since it is important to detect a bleeding condition in the urinary tract at an early stage in its development.
The test for peroxidatively active substances described above is complicated by the presence of ascorbate since this ion is a strong reducing agent which can transfer an electron to the oxidized indicator resulting in false negative results. The inclusion of certain metal ion complexes, such as Fe-HEDTA, in the indicator reagent composition essentially eliminates ascorbate interference, however, the metal ion complexes also demonstrate peroxidase activity thereby catalyzing the color-forming reaction between the peroxide and the oxidizable dye which can, under some circumstances, result in false positives or erroneously high assay results due to additional dye oxidation mediated by the metal ion complex.
The prior art contains numerous references to the wet and dry phase chemistry which can be utilized in assaying fluids for peroxidatively active substances. For example, a wet chemistry assay for a peroxidatively active substance in an acidic medium is presented in R. M. Henry et al., Clinical Chemistry Principles and Techniques, 2nd ed., Harper & Row, pp. 1124-1125 (1974). This wet phase assay procedure employs glacial acetic acid as a buffer, diphenylamine as an indicator dye and hydrogen peroxide. The preferred method of assaying for a peroxidatively active substance involves the use of a dry phase test strip. This is because the test strip format is easier to use in that it requires neither the continual preparation of reagents nor the attendant apparatus.
U.S. Pat. No. 4,587,220 discloses the use of a chelated ferric ion to eliminate ascorbic acid and ascorbate ion interference in an assay for a peroxidatively active substance. This is accomplished by first incorporating a ferric chelate, such as the ferric chelate of N-(2-hydroxyethyl)ethylenediaminetriacetic acid (Fe-HEDTA), into the carrier matrix of a test device. Then, after drying, the indicator dye is incorporated into the carrier matrix. This two-step method of preparing the test device provides an ascorbate resistant test pad that also demonstrates a sufficient stability to resist a false positive assay result during storage.
Yamamoto et al., Int. J. Biol. Macromol., 4, 116-120 (1982) discuss a reduction of ascorbate concentration through auto-oxidation at high pH.
Published European patent application 0253548 describes a liquid composition for determining occult blood with a water-miscible aprotic solvent, water, a chelating agent, a stabilizer, an organic peroxide and an indicator which may be a tetramethylbenzidine derivative or tetramethyl-p-phenylenediamine at a preferred pH range of 10 to 11.
The present invention employs a coupled indicator system that operates at a pH of 10-14 (preferably 12-14) where ascorbate auto-oxidation and/or metal catalyzed oxidation occur at a faster rate thereby tending to reduce ascorbate interference.
In co-pending U.S. application Ser. No. 468,665 there is disclosed an assay for manganese which involves the use of a porphyrin for chelation of the Mn ion and a redox indicator which provides a detectable response when oxidized by oxygen. Suitable redox indicators include a phenylenediamine as developer and a napthol as coupler. A peroxide is not included in this assay thereby reducing potential interference caused by peroxidases in the blood sample being analyzed.